Cloning and sequence analysis of two catechol-degrading gene clusters from the aniline-assimilating bacterium Frateuria species ANA-18

The aniline-assimilating bacterium Frateuria species ANA-18 produced two catechol 1,2-dioxygenases, CD I and CD II, and two muconate cycloisomerases, MC I and MC II. The catA genes catA(1) and catA(2) encoding CD I and CD II, respectively, were cloned from a gene library of this bacterium. The catA(1) gene was clustered with catB(1) encoding MC I, catC(1) encoding muconolactone isomerase (MI), catD encoding beta-ketoadipate enol-lactone hydrolase (ELH), and ORFR1 encoding a putative LysR-type regulator. The organization of these genes was ORF(R1)catB(1)C(1)D. The catA(2) gene also constructed a gene cluster involving catB(2) encoding MC II, catC(2) encoding MI, and ORFR2 encoding a putative LysR-type regulator with the alignment of ORF(R2)catB(2)A(2)C(2). The intergenic regions of ORFR1-catB(1) and ORFR2-catB(2) contained homologous sequences withe catR-catB intergenic region containing a repression binding site and activation binding site of CatR in Pseudomonas putida. These findings suggest that the two cat clusters were regulated independently in their expression. When a product of cloned catD was added to a reaction mixture containing beta-ketoadipate enol-lactone, beta-ketoadipate was produced. This observation showed that the cloned catD encoded ELH and was expressed in Escherichia coli. We found that Frateuria sp. ANA-18 had a large plasmid with a molecular size more than 100 kb. Polymerase chain reaction amplifying partial catA genes and Southern hybridization analyses with probes containing catA genes were conducted, to examine the localization of the two catA genes. We concluded that the catA(1) and catA(2) genes were located on the chromosomal and large plasmid DNAs, respectively, in Frateuria sp. ANA-18. (C) 1999 Elsevier Science B.V. All rights reserved.